Study area
This study was conducted in naturally infected flocks of domestic chickens in Sari city in northern Iran (longitude 51°26′ E, latitude 35°41′ N) from April 2017 to September 2018. Sari is the provincial capital of Mazandaran province, located in the north of Iran, between the northern slopes of the Alborz Mountains and the southern coast of the Caspian Sea (Fig.1). Sari has a humid subtropical climate, with a Mediterranean climate influence. It lies approximately 32m above sea level and has a mild climate characterized by rainfall of 789.2 mm per annum. The mean annual temperatures are 15°C.
Selection of flocks
Out of 45 domestic chicken flocks allocated into the study, 3 flocks were chosen to participate in fecal egg count reduction testing (FECRT) based on flock size. The following criteria were used: (i) the flock had 40 - 60 domestic chickens of different ages and sexes; (ii) anthelmintic drugs had not been used within past eight weeks; (iii) average fecal egg counts (FEC) were higher than or equivalent to 100 eggs per gram (EPG) of feces; and (iv) there was a history of anthelmintic application on the flock in the last three years. When a farmer approved to take part and met the first two principles, fecal samples were collected from twenty randomly selected domestic chickens and tested for FEC. Over 45 flocks were tested to obtain 3 suitable flocks for the FECRT trial.
During the experiment, chickens were kept indoors and fed according to the norms of the field and water was ad lib. Three to four daysbefore the experiment was conducted, feces were examined using the McMaster method, then domestic chickens were randomly assigned to experimental groups with a similar degree of infection to further determine the geometric mean number of eggs per gram of feces.
Fecal egg count reduction test (FECRT)
The FECRT was done for each flock, according to the World Association for the Advancement of Veterinary Parasitology (WAAVP) guidelines for the assessment of anthelmintic efficacy in poultry (14). The ninety infected chickens were randomly distributed into three equal groups of 30 each. Each group was kept in a separate wire-floored cage (550 cm2/chicken).The chickens, naturally infected with helminths, were randomly divided into 3 experimental groups as follows with 10 chickens in each and one group was considered as the control.
Group 1: 5 mg kg−1 body weight (BW) of Fenbendazole for 3 consecutive days
Group 2: 16 mg kg−1 BW of Levamisole
Group 3: placebo; water + DMSO (the control).
Drugs doses were calculated based on a recent study on the efficacy of the Fenbendazole and Levamisole, against internal parasites in chickens [15, 16]. To standardize the doses of Fenbendazole and Levamisole all chickens were treated individually as the doses were given orally via syringe with a blunt needle.
2.3.4. Fecal egg counts (FEC)
Fresh fecal samples were collected from a random sample of chickens (30% of each infected group). Selected chickens were marked, and only on the day of sampling each chicken was placed into an individual cage for defecation. Three grams of fecal samples were collected from the initially selected chickens d 0 (for pre-treatment FEC) and d 14 post-treatment into zip-lock plastic bags, and were kept at 4 °C until processed for FEC using a modified McMaster technique [17]. A decrease in % of FEC describes the percent of reduced FEC between negative control and treated groups [17]. It was calculated on d 14 post-treatment according to the following formula:
% FEC reduction test (FECRT) =100× (FEC control−FEC treated) FEC control
Necropsy
Lastly, for postmortem examination, chickens were euthanized and dissected on day 14 after dosing to obtain worms [18]. Immediately, the gastrointestinal tract of each chicken was removed, and the small intestine was separated. The contents of the intestine was washed into a sieve of 100 µm pore size and inspected for the presence of adult helminths. Subsequently, all visible worms were collected. Evaluation of anthelmintic efficacy was calculated consistent with the guidelines of the World Association for the Advancement of Veterinary Parasitology (WAAVP) for assessing the efficiency of anthelmintics in turkeys and chickens [14]. The efficiency of the treatments was evaluated by comparing FEC in the treated groups and control group.
Statistical analysis
Statistical analysis was performed with SPSS 19.0 software (SPSS Inc, Chicago, IL, USA) with a P value of <0.05 as statistically significant. Repeated measures ANOVA was used to compare the differences between the experimental and control groups of animals.
The effectiveness of anthelmintic drugs was determined by the “control test” method according to the results of coproscopic examinations of animals with the calculation of the geometric mean value of the number of helminth eggs in samples before and 15 days after deworming of animals from the experimental and control groups. The independent samples T-test was uses to compare the efficacy of two anthelmintic drugs.
Interpretation of the FECRT results
Anthelmintic resistance status was understood as suggested by the WAAVP guidelines on AR based on the percentage of fecal egg count reduction (%FECR) and the upper (UCL) and lower (LCL) 95% confidence limits [18]. Therefore, each anthelmintic was confirmed as (i) effective when the %FECR and UCL were both ≥ 95% and the LCL was ≥ 90%, (ii) suspected resistant when %FECR was < 95% or LCL was < 90%, and (iii) ineffective/resistant when both %FECR was < 95% and LCL was < 90%.
Ethical statement
The blinded, randomized, and placebo-controlled study was performed according to the guidance for the experimental study of pharmacological substances. The use of domestic chickens in this study was approved by the Animal Ethics Committee (AEC no. 45403.3.1) of the Ferdowsi University of Mashhad.